Figure 4.

DNA binding protects Stat1 from dephosphorylation. (A) Purified tyrosine-phosphorylated Stat1wt at a concentration of 1 nM was incubated without (–) or with (+) the Stat1-specific tyrosine phosphatase TC45 (PTP) at 30°C for 1 h. Increasing concentrations of a duplex 37-mer DNA (0.5 nM, 5 nM, 25 nM) harboring either a single GAS site (GAS) or an unrelated sequence (Mut) were included in the reaction where indicated. Reaction products were analyzed by Western blotting using anti-P-Stat1. The blots were subsequently reprobed with anti-Stat1 antibody. (B,C) Identical experimental setup as in A, with the indicated Stat1 DNA-binding mutants replacing Stat1wt. (D) Comparison of the effects of single (GAS) or tandem GAS sites (2×GAS) on the dephosphorylation of purified wild-type Stat1. Experimental setup as in A, except that GAS sites were included at concentrations of 0.125 nM, 0.625 nM, and 1.25 nM, respectively.